The study of the recognition or lack of recognition of cancer cells by a host organism has proceeded in many different directions. Understanding of the field presumes some understanding of both basic immunology and oncology.
Early research on mouse tumors revealed that these displayed molecules which led to rejection of tumor cells when transplanted into syngeneic animals. These molecules are "recognized" by T-cells in the recipient animal, and provoke a cytolytic T-cell response with lysis of the transplanted cells. This evidence was first obtained with tumors induced in vitro by chemical carcinogens, such as methylcholanthrene. The antigens expressed by the tumors and which elicited the T-cell response were found to be different for each tumor. See Prehn, et al., J. Natl. Canc. Inst. 18: 769-778 (1957); Klein et al., Cancer Res. 20: 1561-1572 (1960); Gross, Cancer Res. 3: 326-333 (1943), Basombrio, Cancer Res. 30: 2458-2462 (1970) for general teachings on inducing tumors with chemical carcinogens and differences in cell surface antigens. This class of antigens has come to be known as "tumor specific transplantation antigens" or "TSTAs". Following the observation of the presentation of such antigens when induced by chemical carcinogens, similar results were obtained when tumors were induced in vitro via ultraviolet radiation. See Kripke, J. Natl. Canc. Inst. 53: 333-1336 (1974).
While T-cell mediated immune responses were observed for the types of tumor described supra, spontaneous tumors were thought to be generally non-immunogenic. These were therefore believed not to present antigens which provoked a response to the tumor in the tumor carrying subject. See Hewitt, et al., Brit. J. Cancer 33: 241-259 (1976).
The family of tum.sup.- antigen presenting cell lines are immunogenic variants obtained by mutagenesis of mouse tumor cells or cell lines, as described by Boon et al., J. Exp. Med. 152: 1184-1193 (1980), the disclosure of which is incorporated by reference. To elaborate, tum.sup.- antigens are obtained by mutating tumor cells which do not generate an immune response in syngeneic mice and will form tumors (i.e., "tum.sup.+ " cells). When these tum.sup.+ cells are mutagenized, they are rejected by syngeneic mice, and fail to form tumors (thus "tum.sup.- "). See Boon et al., Proc. Natl. Acad. Sci. USA 74: 272 (1977), the disclosure of which is incorporated by reference. Many tumor types have been shown to exhibit this phenomenon. See, e.g., Frost et al., Cancer Res. 43: 125 (1983).
It appears that tum.sup.- variants fail to form progressive tumors because they elicit an immune rejection process. The evidence in favor of this hypothesis includes the ability of "tum.sup.- " variants of tumors, i.e., those which do not normally form tumors, to do so in mice with immune systems suppressed by sublethal irradiation, Van Pel et al., Proc. Natl, Acad. Sci. USA 76: 5282-5285 (1979); and the observation that intraperitoneally injected tum.sup.- cells of mastocytoma P815 multiply exponentially for 12-15 days, and then are eliminated in only a few days in the midst of an influx of lymphocytes and macrophages (Uyttenhove et al., J. Exp. Med. 152: 1175-1183 (1980)). Further evidence includes the observation that mice acquire an immune memory which permits them to resist subsequent challenge to the same tum.sup.- variant, even when immunosuppressive amounts of radiation are administered with the following challenge of cells (Boon et al., Proc. Natl, Acad. Sci. USA 74: 272-275 (1977); Van Pel et al., supra; Uyttenhove et al., supra). Later research found that when spontaneous tumors were subjected to mutagenesis, immunogenic variants were produced which did generate a response. Indeed, these variants were able to elicit an immune protective response against the original tumor. See Van Pel et al., J. Exp. Med. 157: 1992-2001 (1983). Thus, it has been shown that it is possible to elicit presentation of a so-called "tumor rejection antigen" in a tumor which is a target for a syngeneic rejection response. Similar results have been obtained when foreign genes have been transfected into spontaneous tumors. See Fearson et al., Cancer Res. 48: 2975-1980 (1988) in this regard.
A class of antigens has been recognized which are presented on the surface of tumor cells and are recognized by cytotoxic T cells, leading to lysis. This class of antigens will be referred to as "tumor rejection antigens" or "TRAs" hereafter. TRAs may or may not elicit antibody responses. The extent to which these antigens have been studied, has been via cytolytic T cell characterization studies in vitro i.e., the study of the identification of the antigen by a particular cytolytic T cell ("CTL" hereafter) subset. The subset proliferates upon recognition of the presented tumor rejection antigen, and the cells presenting the antigen are lysed. Characterization studies have identified CTL clones which specifically lyse cells expressing the antigens. Examples of this work may be found in Levy et al., Adv. Cancer Res. 24: 1-59 (1977); Boon et al., J. Exp. Med. 152: 1184-1193 (1980); Brunner et al., J. Immunol. 124: 1627-1634 (1980); Maryanski et al., Eur. J. Immunol. 124: 1627-1634 (1980); Maryanski et al., Eur. J. Immunol. 12: 406-412 (1982); Palladino et al., Canc. Res. 47: 5074-5079 (1987). This type of analysis is required for other types of antigens recognized by CTLs, including major histocompatibility antigens, the male specific H-Y antigens, and a class of antigens, referred to as "tum.sup.- " antigens, and discussed herein.
A tumor exemplary of the subject matter described supra is known as P815. See DePlaen et al., Proc. Natl. Acad. Sci. USA 85: 2274-2278 (1988); Szikora et al., EMBO J 9: 1041-1050 (1990), and Sibille et al., J. Exp. Med. 172: 35-45 (1990), the disclosures of which are incorporated by reference. The P815 tumor is a mastocytoma, induced in a DBA/2 mouse with methylcholanthrene and cultured as both an in vitro tumor and a cell line. The P815 line has generated many tum.sup.- variants following mutagenesis, including variants referred to as P91A (DePlaen, supra), 35B (Szikora, supra), and P198 (Sibille, supra). In contrast to tumor rejection antigens--and this is a key distinction--the tum.sup.- antigens are only present after the tumor cells are mutagenized. Tumor rejection antigens are present on cells of a given tumor without mutagenesis. Hence, with reference to the literature, a cell line can be tum.sup.+, such as the line referred to as "P1", and can be provoked to produce tum.sup.- variants. Since the tum.sup.- phenotype differs from that of the parent cell line, one expects a difference in the DNA of tum.sup.- cell lines as compared to their tum.sup.+ parental lines, and this difference can be exploited to locate the gene of interest in tum.sup.- cells. As a result, it was found that genes of turn variants such as P91A, 35B and P198 differ from their normal alleles by point mutations in the coding regions of the gene. See Szikora and Sibille, supra, and Lurquin et al., Cell 58: 293-303 (1989). This has proved not to be the case with the TRAs of this invention. These papers also demonstrated that peptides derived from the tum.sup.- antigen are presented by the L.sup.d molecule for recognition by CTLs. P91A is presented by L.sup.d, P35 by D.sup.d and P198 by K.sup.d.
Prior patent application PCT/US92/04354, and U.S. Pat. No. 5,342,774, both of which are incorporated by reference describe inventions involving, inter alia, genes and other nucleic acid molecules which code for various TRAPs, which are in turn processed to tumor rejection antigen, or "TRAs". SEQ ID NOS: 1-26 which are a part of the subject PCT application, present sequences of genes coding for various TRAPs, and the TRA referred to hereafter as MZ2-E, which is derived from MAGE-1 TRAP (SEQ ID NO: 26).
The genes are useful as a source for the isolated and purified tumor rejection antigen precursor and the TRA themselves, either of which can be used as an agent for treating the cancer for which the antigen is a "marker", as well as in various diagnostic and surveillance approaches to oncology, discussed infra. It is known, for example, that tum.sup.- cells can be used to generate CTLs which lyse cells presenting different tum.sup.- antigens as well as tum.sup.+ cells. See, e.g., Maryanski et al., Eur. J. Immunol 12: 401 (1982); and Van den Eynde et al., Modern Trends in Leukemia IX (June 1990), the disclosures of which are incorporated by reference. The tumor rejection antigen precursor may be expressed in cells transfected by the gene, and then used to generate an immune response against a tumor of interest.
In the parallel case of human neoplasms, it has been observed that autologous mixed lymphocyte-tumor cell cultures ("MLTC" hereafter) frequently generate responder lymphocytes which lyse autologous tumor cells and do not lyse natural killer targets, autologous EBV-transformed B cells, or autologous fibroblasts (see Anichini et al., Immunol. Today 8: 385-389 (1987)). This response has been particularly well studied for melanomas, and MLTC have been carried out either with peripheral blood cells or with tumor infiltrating lymphocytes. Examples of the literature in this area including Knuth et al., Proc. Natl. Acad. Sci. USA 86: 2804-2802 (1984); Mukherji et al., J. Exp. Med. 158: 240 (1983); Herin et all, Int. J. Canc. 39: 390-396 (1987); Topalian et al, J. Clin. Oncol 6: 839-853 (1988). Stable cytolytic T cell clones have been derived from MLTC responder cells, and these clones are specific for the tumor cells. See Mukherji et al., supra, Herin et all, supra, Knuth et al., supra. The antigens recognized on tumor cells by these autologous CTLs do not appear to represent a cultural artifact, since they are found on tumor cells in vivo. Topalian et al., supra; Degiovanni et al., Eur. J. Immunol. 20: 1865-1868 (1990). These observations, coupled with the techniques used herein to isolate the genes for specific murine tumor rejection antigen precursors, have led to the isolation of nucleic acid sequences coding for tumor rejection antigen precursors of TRAs presented on human tumors. It is now possible to isolate the nucleic acid sequences which code for tumor rejection antigen precursors, including, but not being limited to those most characteristic of a particular tumor, with ramifications that are described infra.
Additional work has focused upon the presentation of TRAs by the class of molecules known as major histocompatibility complexes, or "MHCs". Human forms of these molecules are "human leukocyte antigens" or "HLAs". This work has resulted in several unexpected discoveries regarding the field. Specifically, U.S. Pat. Nos. 5,405,940 and 5,462,871, the disclosures of which is incorporated by reference, nonapeptides are taught which are presented by HLA-A1 molecules. The reference teaches that given the known specificity of particular peptides for particular HLA molecules, one should expect particular peptides to bind one HLA molecule. These peptides are also presented in Traversari et al., J. Exp. Med. 176: 1453-1457 (1992). This is important, because different individuals possess different HLA phenotypes. As a result, while identification of particular peptides or of particular motifs, and the peptides which are members thereof, as being partners for a specific HLA molecule has diagnostic and therapeutic ramifications, these are only relevant for individuals with that particular HLA phenotype. There is a need for further work in the area, because cellular abnormalities are not restricted to one particular HLA phenotype, and targeted therapy requires some knowledge of the phenotype of the abnormal cells at issue.
In U.S. patent application Ser. No. 08/008,446, filed Jan. 22, 1993, now abandoned and incorporated by reference, the fact that the MAGE-1 expression product is processed to a second TRA is disclosed. This second TRA is presented by HLA-Cw* 1601 molecules. The disclosure shows that a given TRAP can yield a plurality of TRAs.
In U.S. Pat. No. 5,487,974 and incorporated by reference herein, tyrosinase is described as a tumor rejection antigen precursor. This is a well known molecule as per Kwon, U.S. Pat. No. 4,898,814. This reference discloses that a molecule which is produced by some normal cells (e.g., melanocytes), is processed in tumor cells to yield a tumor rejection antigen that is presented by HLA-A2 molecules. The peptide presented thereby is described in U.S. Application Ser. No. 08/057,714, filed Apr. 28, 1993, now abandoned also incorporated by reference. Additional tyrosinase derived peptides presented by HLA molecules are set forth in Ser. No. 08/203,054, now U.S. Pat. No. 5,530,096 and Ser. No. 08/233,305 now U.S. Pat. No. 5,519,117 filed Feb. 28, 1994 and Apr. 26, 1994 and are incorporated by reference.
Other peptides which are TRAs are described in additional patent applications. U.S. patent application Ser. No. 08/195,186, now U.S. Pat. No. 5,558,995 filed Feb. 14, 1994, and incorporated by reference herein, sets forth three peptides, which are derived from MAGE-1 and which complex with HLA-Cw* 1601. Ser. No. 08/196,630, now abandoned filed Feb. 15, 1994, discloses an unrelated tumor rejection antigen precursor, the so-called "BAGE" gene, and peptides derived therefrom, which are processed and then presented by HLA-Cw* 1601. Additional coding sequences for a tumor rejection antigen precursor referred to as Melan-A are set forth in Ser. No. 08/032,978, now U.S. Pat. No. 5,620,886 filed Mar. 18, 1993 and incorporated by reference. A more extended sequence for this gene is set forth in Ser. No. 08/272,351, now abandoned filed Jul. 8, 1994 incorporated by reference. In Ser. No. 08/96,039, filed Jul. 22, 1993, now abandoned the sequence of tumor rejection antigen precursor GAGE is set forth, and is incorporated by reference.
A series of peptides which provoke lysis by cytolytic T cells when presented by MHC molecules are set forth in Ser. No. 08/217,186, now U.S. Pat. No. 5,585,461 Ser. No. 08/217,188, now U.S. Pat. No. 5,554,724 and Ser. No. 08/217,187, now U.S. Pat. No. 5,554,506 all filed on Mar. 24, 1994, and all of which are incorporated by reference herein. The first of these applications discloses MAGE-3 derived peptides presented by HLA-A2. Five peptides are of interest. The second application presents 11 sequences derived from MAGE-2, believed to complex with HLA-A2.1 molecules. The last of these applications discloses two additional peptides derived from MAGE-3 which complex to HLA-A2. Ser. No. 08/190,411, now U.S. Pat. No. 5,541,104 filed Apr. 1, 1994 and incorporated by reference, sets forth three peptides derived from MAGE-1, which are immunogenic in that they provoke production of antibodies in a host animal to which they have been administered. Ser. No. 08/253,503, now U.S. Pat. No. 5,589,334 filed Jun. 3, 1994 and incorporated by reference, teaches a further tumor rejection antigen precursor gene and a peptide, derived therefrom, which is presented by HLA-B44 molecules. Further in the application of Coulie, Ikeda and Boon-Falleur, Ser. No. 08/316,231 now U.S. Pat. No. 5,830,753 incorporated by references, a sequence coding for a tumor rejection antigen precursor known as DAGE is set forth. DAGE is found almost universally on tumor cells, and only on testis cells with respect to normal cell expression. This makes it especially useful for cancer diagnosis and in the applications disclosed herein. The above listing should not be presumed to be exhaustive of the TRAP and TRA literature, but is presented to show its diversity and the fact that these materials not only provoke T cell proliferation, but also stimulate production of antibodies. It is well known that antibody producing cells can be used as a source to produce hybridomas, which in turn produce monoclonal antibodies. Thus, when the term "antibodies" is used herein, it encompasses both polyclonal and monoclonal antibodies.
U.S. patent application Ser. No. 08/142,368 now U.S. Pat. No. 5,925,729, Ser. No. 08/190,411 now U.S. Pat. No. 5,541,104 and Ser. No. 08/315,961, now abandoned all incorporated by reference discuss the usefulness of combining TRAPs or TRAs with various materials as adjuvants, to produce vaccines, immunogenic compositions, etc. Adjuvants, broadly defined, are substances which promote immune responses. Frequently, the adjuvant of choice if Freund's complete adjuvant, or killed B. pertussis organisms, used in combination with alum precipitated antigen. A general discussion of adjuvants is provided in Goding, Monoclonal Antibodies: Principles & Practice (Second edition, 1986), at pages 61-63, which are incorporated by reference herein. Goding notes, however, that when the antigen of interest is of low molecular weight, or is poorly immunogenic, coupling to an immunogenic carrier is recommended. Such molecules, according to Godinq, generally have molecular weights below about 1000. Among the carriers suggested by Goding, at page 283, are keyhole limpet hemocyanin, bovine serum albumin, ovalbumin, and fowl immunoglobulin.
What is problematic about such carriers, however, is that frequently they are also immunogenic themselves. Thus, the immune response may be a general one, with part, most, or all of it being directed against the carrier molecule rather than the immunogen itself.
Exemplary of developments in the art as they relate to adjuvants is U.S. Pat. No. 5,057,540 to Kensil, et al, the disclosure of which is incorporated by reference herein. Kensil et al disclose the preparation of various saponin extracts, which are useful as adjuvants in immunogenic compositions. As natural products, the extracts are not completely defined. Kensil, et al do provide a complete and enabling disclosure for how various extracts, including QA-7, QA-19, and QA-21 (also referred to as QS-21) are prepared. Experiments are set forth in which bovine serum albumin ("BSA") was combined with various extracts (examples 8 and 9), and where feline leukemia virus recombinant glycoprotein "gp7OR.DELTA. was tested, following absorption to aluminum hydroxide (alum). The two immunogens tested, however, are expected to be immunogenic in their own right (gp7OR.DELTA. has a molecular weight of 70 kd, and serum albumin has about the same molecular weight). No experiments were carried out at all on molecules which should, per se, be considered to be poorly or even non-immunogenic, and thus would be expected to require the use of alum absorption or the use of haptenic carriers for provocation of a response.
In PCT Application WO9219758, which corresponds to defensive publication 7697275, which is incorporated by reference herein, an adjuvant referred to as "MTP-MF59" is disclosed. This adjuvant is used in connection with a Plasmodium falciparum protein, "Pfs-25-B". This combination is described as a transmission blocking vaccine. The P. falciparum protein is itself large enough to be immunogenic. Thus, none of the art shows that the improved adjuvants can be used in combination with presumptively non-immunogenic proteins and peptides to yield immunologically effective compositions. This is especially true for TRAP and TRA molecules, as outlined supra.
Granulocyte-macrophage colony stimulating factor ("GM-CSF" hereafter), is a well known cytokine, having a molecular weight of about 18-32 kDA on SDS-PAGE, or 30 kDA by gel filtration. It contains 127 amino acids. Other properties of the molecule are summarized in, e.g. Crosier et al., "Granulocyte-Macrophage Colony Stimulating Factor" in Aggarwal, et al., Human Cytokines: Handbook For Basic And Clinical Research (Blackwell Scientific Publications, 1992), chapter 14 in particular, the disclosure of which is incorporated by reference. The patent literature on GM-CSF is vast. Exemplary of this literature are U.S. Pat. Nos. 5,437,994; 5,211,947; 5,199,942; 5,198,417; 5,178,855; and 5,162,111, all of which are incorporated by reference. These patents disclose various uses of GM-CSF in the area of treatment of particular diseases and pathologies, where expansion of the granulocyte and/or macrophage population of the recipient is desirable or necessary. None of these references is desirable or necessary. None of these references teach the use of GM-CSF as an adjuvant, in the sense provided by the preceding example. It has now been found that GM-CSF does possess properties which permit it to be used as an adjuvant to improve, enhance, or provoke an immune response against a particular immunogen. This is the subject of the invention, described in greater detail infra.